Mouse gestation takes place over 20 days. Germ cells, first detected in mouse embryos at day 7.25, migrate from the extra-embryonic allantois to the genital ridge where the primitive gonad differentiates into an ovary by day 13 in XX females. Using high through-put sequencing and modern informatics to screen urogenital cDNA libraries, we have identified a novel gene located on the X chromosome. This gene, designated Nt2, is expressed preferentially in the somatic cells of female gonads at the time of sex determination. The resultant protein contains a homeobox domain near its carboxyl terminus raising the possibility that NT2 may play a role in gonadogenesis. The gene has been successfully targeted in embryonic stem cells that are currently being used to establish mouse lines for analysis of the null phenotype.At birth the ovary contains its full complement of germ cells, each surrounded by a single layer of granulosa cells which together form the primordial follicles. We have identified a novel, oocyte-specific, basic helix-loop-helix transcription factor, FIG (Factor In the Germline, alpha). The single-copy Fig gene has been disrupted in embryonic stem cells and these cells have been used to establish mouse lines. Female mice lacking FIG are sterile because of germ cell depletion secondary to an inability to form primordial follicles perinatally. Identification of downstream targets of FIG should provide additional insights into the molecular basis of follicle formation. After the onset of folliculogenesis, FIG also modulates the oocyte-specific expression of the single-copy genes that encode ZP1, ZP2 and ZP3 (i.e., zona transcripts are not detected in Fig null mice). Normally, the three zona glycoproteins are secreted during folliculogenesis to form the zona pellucida, an extracellular matrix that mediates order-specific sperm binding to the egg (e.g., human sperm will not bind to the mouse zona pellucida). Using transgenesis, we have determined that mouse ZP1 is not required for formation of the zona pellucida, order-specific sperm binding or fertility. In contrast, both ZP2 and ZP3 are required for matrix formation without which mice are infertile. Although the replacement of either mouse ZP2 or mouse ZP3 with their human homologue restores the zona pellucida matrix, it does not affect the specificity of sperm binding (i.e., mouse, but not human, sperm bind and fertilize eggs enclosed in humanized zonae pellucidae). Breeding studies to establish mouse lines expressing both human ZP2 and ZP3 or mutant forms of the mouse zona proteins are being pursued to further investigate the molecular basis of sperm-egg interactions. - zona pellucida, fertilization, sex determination, oocyte-specific gene expression, factor in the germline,